Overexpression of prostate specific membrane antigen by canine hemangiosarcoma cells provides opportunity for the molecular detection of disease burdens within hemorrhagic body cavity effusions

Abstract

Background: Canine hemangiosarcoma (cHSA) is a highly metastatic mesenchymal cancer that disseminates by hematogenous and direct implantation routes. Therapies for cHSA are generally ineffective, in part due to advanced clinical disease stage at the time of diagnosis. The validation of conventional molecular methods for detecting novel biomarkers preferentially expressed by cHSA could lead to more timely diagnosis, earlier therapeutic interventions, and improved outcomes. In humans, prostate-specific membrane antigen (PSMA) is a transmembrane protein overexpressed by prostate carcinoma and tumor-associated endothelium of various solid cancer histologies. Importantly, the preferential overexpression of PSMA by certain cancers has been leveraged for the development of diagnostic molecular imaging reagents and targeted therapeutics. Recently, PSMA has been qualitatively demonstrated to be expressed in cHSA cell lines, however, quantitative PSMA expressions and the potential utility of PSMA transcript identification in biologic fluids to support the presence of microscopic cHSA burden has not been reported. Therefore, this study sought to characterize the differential quantitative expressions of PSMA between cHSA and non-malignant tissues, and to determine the potential diagnostic utility of PCR-generated PSMA amplicons as a surrogate of rare cHSA cells dwelling within peritoneal and pericardial cavities.

Methods: Quantitative gene and protein expressions for PSMA were compared between one normal endothelial and six cHSA cell lines by RT-PCR, western blot analysis, and fluorescent microscopy. Additionally, gene and protein expressions of PSMA in normal canine tissues were characterized. Graded expressions of PSMA were determined in spontaneously-arising cHSA tumor samples and the feasibility of qualitative PCR as a molecular diagnostic to detect PSMA transcripts in whole blood from healthy dogs and hemorrhagic effusions from cHSA-bearing dogs were evaluated.

Results: PSMA gene and protein expressions were elevated (up to 6-fold) in cHSA cells compared with non-malignant endothelium. By immunohistochemistry, protein expressions of PSMA were detectable in all cHSA tissue samples evaluated. As predicted by human protein atlas data, PSMA's expression was comparably identified at substantial levels in select normal canine tissues including kidney, liver, and intestine. In young healthy pet dogs, PSMA amplicons could not be identified in circulating whole blood yet were detectable in hemorrhagic effusions collected from pet dogs with confirmed cHSA or PSMA-expressing cancer.

Conclusions: PSMA is quantitatively overexpressed in cHSA compared to normal endothelium, but its protein expression is not restricted to only cHSA tumor tissues, as specific visceral organs also substantively express PSMA. Optimized qualitative PCR methods failed to amplify PSMA amplicons sufficiently for visible detection in circulating whole blood derived from healthy young dogs, yet PSMA transcripts were readily identifiable in hemorrhagic effusions collected from pet dogs with histologically confirmed cHSA or PSMA-expressing cancer. While preliminary, findings derived from a limited cohort of normal and diseased pet dogs provocatively raise the potential value of PSMA amplicon detection as an ancillary molecular diagnostic test for supporting the presence of microscopic cHSA disease burden within hemorrhagic body cavity effusions.

Fig 1. Gene transcription of PSMA across cell lines.

(A) Validation of canine-specific TaqMan PSMA primer efficiency across 5-log orders of RNA concentration using CPA, a canine prostatic carcinoma cell line. (B) Comparative PSMA gene expression across canine cell lines. Relative PSMA expressions for six cHSA cell lines (red) compared to the non-malignant canine endothelial cell line, CAoEC (green). Positive and negative control cell lines include CPA (blue) and ACE-1 (purple), respectively. Representative data presented from 3 independent biologic replicates with 5 technical replicates for each cell line.

Fig 2. Antibody validation for canine PSMA protein expression.

Validation of antibodies for detecting PSMA in human and canine cell lines by (A) confocal fluorescent microscopy, and (B) western blot analysis. Positive and negative controls for canine and human include (CPA and ACE-1) and (LNCaP and PC3), respectively.

Fig 3. Comparative PSMA protein expressions across cell lines.

Comparison of PSMA protein expressions and associated histograms for six cHSA cell lines (red) relative to the non-malignant CAoEC line (green) by (A and B) western blot analysis, and (C and D) by confocal fluorescent microscopy. Canine positive and negative controls are CPA (blue) and ACE-1 (purple), respectively. Significance p < 0.05 and p < 0.01 denoted by “*” and “**”, respectively.

Fig 4. Characterization of PSMA in cHSA tumor samples.

Spontaneously-arising cHSA tumor samples involving the spleen, liver, and lung tissues, with representative (A) histology by H&E and PSMA immunohistochemistry (Score 3; magnification 400x), and comparison (B) of staining properties for cHSA by CD31 and PSMA (magnification 100x).

Fig 5. Optimization of qualitative PCR methodology and dynamic range of detection for PSMA.

(A) Differential PSMA amplicon generation by qualitative PCR with the lowly expressing PSMA cHSA cell line (FITZ; red) compared with CAoEC (green) across different thermocycles ranging from 24 to 30. Determination of qualitative PCR sensitivity for detecting PSMA amplicons at 27 cycles with a logarithmic titration of (B) FITZ cells only or (C) when FITZ cells are spiked into whole blood. Lowest cell density capable that produces faintly visible amplicons is 10⁴ FITZ cells, alone or in addition to whole blood nucleated cells. Amplicon production expressed as a ratio of PSMA/β actin pixels/area. Abbreviations FITZ (FZ; red) and CAoEC (CE; green).

Fig 6. Tandem cytologic evaluation and PSMA amplicon generation from hemorrhagic effusions.

(A) Cytologic characterization of hemorrhagic effusions collected from five dogs, three with hemoabdomen (Dogs 1, 2, and 4) and two with hemorrhagic pericardial effusion (Dogs 3 and 5). Note the identification of atypical cells in Dogs 1–3 and 5 (right, top panel). (B) Generation of visible PSMA amplicons (27 cycles) from hemorrhagic effusions overtly positive for Dogs 1–3 and 5, and weakly positive for Dog 4. (C) Immunohistochemical evaluation of primary tumors from Dogs 1–3 and 5, confirming that primary tumors presumed to be the source of exfoliative cHSA cells stain positively for PSMA.

Fig 7. Generation of PSMA amplicons from non-cHSA tumor histology.

Immunohistochemical evaluation of tumor from Dog 4, showing immunopositivity for vimentin, neuron-specific enolase, and PSMA only. Dotted black line separates normal liver tissue (right) from tumor tissue (left). CD18 and CD31 immunohistochemistry fail to demonstrate immunoreactivity within tumor cells, but do stain resident macrophages and normal blood vessels, respectively. Magnification 400x; abbreviations CK, cytokeratin; VM, vimentin; NSE, neuron-specific enolase.